We have developed a novel hybrid vector system consisting of human L1 retrotransposons with associated transgene cassettes delivered by a helper-dependent ("gutted") adenovirus vector. This hybrid vector transduces cells by a two-stage mechanism: in the first (adenovirus) stage, the helper-dependent adenovirus serves as a carrier for efficient delivery and transient expression of its encoded L1/transgene cassette, and in the second (retrotransposon) stage, the L1 retroelement and its associated transgene then permanently integrate into the genome of the adenovirus-transduced cells. We propose that this novel retrotransposon-adenovirus hybrid vector system will be useful both as a vehicle for efficient delivery and long-term stable transduction of therapeutic genes, and as a tool to elucidate aspects of retrotransposon biology that have previously been difficult to study. We now propose to construct and test second-generation retrotransposon-adenovirus hybrid vectors that contain a newly isolated L1 retrotransposon, L1RP, exhibiting the highest transposition frequency to date. We will initially test a series of L1RP-based hybrid constructs in cell culture studies to optimize vector-associated parameters that may affect retrotransposition efficiency, and in particular we will investigate the use of the highly prostate-specific probasin promoter as a novel mechanism to regulate retrotransposition and thereby enhance the selectivity and safety of this vector system. We will also utilize these vectors to study host cell-associated parameters, such as cell cycle and double-strand break repair, that may also affect retrotransposition efficiency, and thereby seek to contribute to our understanding of the mechanisms involved in L1 retrotransposition. We will also investigate the in vivo efficiency and durability of transduction mediated by optimized retrotransposon-adenovirus hybrid vectors. To explore the potential of these hybrid vectors for use in therapeutic applications, we will test their ability to achieve efficient in vivo gene delivery and long-term expression of an anti-angiogenic factor, endostatin, after intra- tumoral as well as systemic administration in animal models of prostate cancer.